I am working on neural differentiation of dental pulp stem cells (DPSC). I am checking some ion channels that play role in the differentiation and want to do an absolute quantification of my qPCR results.I am using undifferentiated DPSC to generate my standard curve. I came across a problem where my undifferentiated DPSC doesn't express specific genes and are expressed only by differentiated cells.
In this case I thought of 2 options
1: Can use some other cells that are strongly positive for our gene of interest. I am not sure, if adding another cell type would bring in another factor leading to error? And if I can compare two genes that uses different cells type to generate standard curve?
2. Can use DPSC and differentiate the cells and use them when we know they express the gene of interest. This is gamble because we need to exactly know when the cells express the gene the most.
Could someone kindly advise me a possible solution for the above problem.
Thank you
Typically, you would CLONE your gene of interest (or at least a portion of it), and use the linearized plasmid DNA to create your standard curve.
Good luck!
Thank you Dr. Clark for your suggestion. Could you pls suggest any other possible solution. I m in in final semester of phd. So need a quickly workable solution.
Try to clone the sequence of your gene and transfer it inside the vector. You obtain more copies of it by cloning and after the quantification of them, prepare your standard curve at different dilution.
Another thing. Are you sure that the protocol of your qPCR is optimase?
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